Pasteurella haemolytica, P. multocida, and Haemophilus somnus are members of the Family Pasteurellaceae. Each is involved in respiratory disease syndromes in domestic cattle. These organisms have proven difficult to genetically manipulate, and therefore the construction of live, attenuated vaccines has been hampered.
Live attenuated bacterial strains generally provide superior protection as compared to killed bacterial vaccines (bacterins). In general, live vaccines elicit a stronger cell mediated response in the host than do bacterins. The superior immunity provided by attenuated live organisms may be explained by their ability to induce expression of stress-proteins and, possibly, of certain toxins within the host. The immune response generated by live organisms can be directed against these abundant proteins and thereby provide better protection.
There is a need in the art for live, attenuated vaccines against respiratory disease syndrome in domestic cattle caused by the Pasteurellaceae. There is also a need for techniques and tools to facilitate the construction of such vaccines.
It is an object of the invention to provide a replication-conditional plasmid.
It is another object of the invention to provide a cell-free preparation of a plasmid which is purified from genomic DNA and which is replication-conditional.
It is still another object of the invention to provide Pasteurellaceae host cells which comprise a plasmid which is replication-conditional.
It is an object of the invention to provide methods for introducing DNA into H. somnus.
It is an object of the invention to provide methods for mutagenizing H. somnus.
It is an object of the invention to provide H. somnus transformant strains.
It is an object of the invention to provide H. somnus mutant strains.
It is another object of the invention to provide genetically engineered H. somnus.
It is an object of the invention to provide a method of introducing a DNA segment into a Pasteurellaceae genome.
It is another object of the invention to provide genetically modified Pasteurellaceae.
These and other objects of the invention are provided by one or more embodiments described below. In one embodiment a plasmid which is conditional for replication in H. somnus, P. multocida, and P. haemolytica is provided.
In another embodiment of the invention a cell-free preparation of plasmid DNA is provided. The plasmid DNA is purified free of genomic DNA. The plasmid is temperature-conditional for replication in H. somnus, P. multocida, and P. haemolytica.
In another embodiment of the invention a host cell of the family Pasteurellaceae is provided. The host cell comprises a plasmid which is replication-conditional in H. somnus, P. multocida, and P. haemolytica.
In one embodiment of the invention, a method of introducing DNA to H. somnus is provided. The method comprises the steps of: providing a DNA molecule; methylating the DNA molecule with a methyl transferase enzyme having a recognition site of 5xe2x80x2-GCGC-3xe2x80x2, to form methylated DNA; and transforming H. somnus cells with the methylated DNA.
In another embodiment of the invention a method for producing a mutation in a particular region of DNA of an H. somnus genome is provided. The method comprises the steps of: isolating a region of the genome from H. somnus; introducing a mutation into the region to form a mutated DNA region; methylating the mutated DNA region with a methylating enzyme which inhibits endonuclease cleavage at a recognition sequence 5xe2x80x2-GCGC-3xe2x80x2 to form methylated DNA; introducing the methylated DNA into an H. somnus cell to form transformants; and screening the transformants for those which have the mutation in the region on chromosomal DNA of the H. somnus cell.
In yet another embodiment of the invention a preparation is provided of an isolated HsoI restriction endonuclease.
In still other embodiments H. somnus mutants and transformants made by the process of the invention are provided.
In another embodiment of the invention a method is provided of introducing a DNA segment to a Pasteurellaceae genome comprising: administering to a Pasteurellaceae cell a recombinant construct comprising the DNA segment and a plasmid which is temperature-conditional for replication in the Pasteurellaceae cell to form transformants; subjecting the transformants to a non-permissive temperature; screening the transformants for the presence of the DNA segment; and screening the transformants for the absence of the plasmid.
In yet another embodiment a genetically modified Pasteurellaceae is provided which is made by the method of administering to a Pasteurellaceae cell a recombinant construct comprising the DNA segment and a plasmid which is temperature-conditional for replication in the Pasteurellaceae cell to form transformants; subjecting the transformants to a non-permissive temperature; screening the transformants for the presence of the DNA segment; and screening the transformants for the absence of the plasmid.
These and other embodiments of the invention provide the art with the means to construct desirable mutants and transformants of the economically important and previously intractable Pasteurellaceae family of pathogens.